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Validation of Analytical Method :

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Definition:
Method validation is the process of proving that ananalytical method is acceptable for its intended purposes.
METHOD VALIDATION = ERROR ASSESSMENT

Why Method Validation is Important? :

Why Method Validation is Important? Develops confidence in using the method & Proof thatMethod is suitable for its intended purpose,
The purpose of analytical measurement is to get consistent, reliable and accurate data.
Incorrect measurement results can lead to tremendous costs.
Regulatory requirement, Equal importance for those working in a regulated and in an accredited environment.
U.S. FDA, ISO etc.

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When to be validated?
Partial validation after development of method.
Complete validation after manufacturing formula is finalized.
Which methods are to be validated
Compendial: Pharmacopoeial method
Verification of suitability of method
Non compendial methods: Laboratory developed methods.
Pharmacopoeial methods used outside its scope.

Specificity :

Specificity Ability of an analytical method to measure the analyte free from interference due to other components. Selectivity
Bias

Specificity: ICH/USP :

Specificity: ICH/USP The ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the matrix
The degree of interference
Active Ingredients
Excipients
Impurities (synthetic precursors, enantiomers)
Degradation Products
Placebo Ingredients

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Combination of 2 or more analytical procedures may be required to achieve necessary level of discrimination (i.e. LC-MS, etc.)
Stability indicating analytical methods should always be specific
Analysts should ascertain whether the peaks within a sample chromatogram are pure or consist of more than one compound. Therefore should know how many compounds are in the sample or use procedures to detect peak purity

Linearity :

Linearity Ability of an assay to elicit a direct and proportional response to changes in analyte concentration.

Contd…. :

Contd…. The Ability of the method to obtain test results that are directly proportional to concentration within a given range
Method: dilution of stock solution/separate weightings
Expressed as the variance of the slope of the regression line
Correlation coefficient, y-intercept, slope of regression line and residual sum of squares should be presented together with plot of the data

Range :

Range The interval between the upper and lower concentrations of analyte in the sample that have been demonstrate to have a suitable level of precision, accuracy, and linearity.

Range :

Range Normally derived from Linearity studies.
Established by confirming that the method provides acceptable degree of linearity, accuracy, and precision.
Specific range dependent upon intended application of the procedure.

Accuracy :

Accuracy Closeness of the test results obtained by the method to the true value.

Precision :

Precision The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample.
Should be investigated using homogeneous, authentic samples. Strike Strike Strike Strike Strike Strike Ball Ball Ball Ball Ball Ball Ball Ball

Accuracy Vs Precision :

Precision… Considered at 3 Levels :

Repeatability :

Repeatability Express the precision under the same operating conditions over a short interval of time.
Also referred to as Intra-assay precision

Intermediate Precision :

Intermediate Precision Express within-laboratory variations.
Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval.
Known as part of Ruggedness in USP (Different Analysts, Different Laboratories, Different Instruments, Different Reagents, Different Days) Depends on the circumstances under which the procedure is intended to be used.
Studies should include varying days, analysts, equipment, etc.

Reproducibility :

Definition: Ability to reproduce data within the predefined precision
Determination: SD, RSD and confidence interval
Repeatability test at two different labs. Reproducibility

Reproducibility Study :

Reproducibility Study

Detection Limit (DL) :

Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.
Estimated by Signal to Noise Ratio of 3:1. Detection Limit (DL) Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision.
Estimated by Signal to Noise Ratio of 10:1. Quantitation Limit (QL)

LOD, LOQ and SNR :

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Definition: Capacity to remain unaffected by small but deliberate variations in method parameters
Determination: Comparison results under differing conditions with precision under normal conditions
Variations may include: stability of analytical solution, variation of pH in a mobile phase, different column (lot/supplier), temperature, flow rate. Robustness

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Solutes may readily decompose prior to chromatographicinvestigations e.g. during sample preparation, extraction,cleanup, phase transfer or storage of prepared vials(refrigerators or automatic sampler).
Method development should investigate the stability of theAnalytes AND standards. Stability of analytical solution System stability
Stability of the samples being analyzed in a sample solution. e.g. 1 – 48 hours using a single solution.
should be determined by replicate analysis of the samplesolution.

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The checking of a system, before or during analysis of unknowns, to ensure system performance.
“No sample analysis is acceptable unless the requirements for system suitability have been met.” (USP Chapter 621)
Plate Count, Tailing, Resolution
Determination of reproducibility (%RSD)
For %RSD < 2.0%, Five replicates
For %RSD > 2.0%, Six replicates
System Suitability "Sample“ - A mixture of main components andexpected by-products utilized to determine system suitability
“Whenever There is a Significant change in Equipment or ReagentsSystem Suitability Testing Should be Performed” (USP Chapter 621) SYSTEM SUITABILITY

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Change in the analytical procedure, drug substance, drug product, the changes, may necessitate revalidation of the analytical procedures.
“The degree of revalidation depends on the nature of the change.”
“FDA intends to provide guidance in the future on post-approval changes in analytical procedures.” Revalidation should accompany
formulation changes (new samples with new compounds or new matrices)
manufacturing batch changes
new analysts with different skills,
new instruments with different characteristics,
new location with different environmental conditions,
new chemicals and/or reference standards and
modification of analytical parameters. Revalidation

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General requirements Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample integrity
Change control (e.g., synthesis, FPP composition) Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2 (R1)